Project Summary Chromatin organization in the nucleus is highly regulated to provide precise control over gene expression. The most dramatic example of the relationship between chromatin structure and gene regulation is X Chromosome Inactivation (XCI). XCI epigenetically converts one female X chromosome into a transcriptionally silent inactive X (Xi) to equalize dosage between males and females. XCI is initiated by spreading of the long non-coding RNA Xist in cis which re-organizes the Xi into a compact, bipartite structure, and targets the Xi to the nuclear periphery. Maintenance of the Xi must be achieved to prevent aberrant expression of X-linked genes, which is associated with autoimmune diseases and cancers. However, how silencing or nuclear organization of the Xi is regulated during maintenance of XCI remains to be well understood. The current paradigm of maintenance is stable association of Xist RNA with the Xi in all female somatic cells. Recently, the Anguera lab found a novel mechanism of XCI in female lymphocytes. In nave female B cells, Xist RNA is not localized to the Xi, but surprisingly, after stimulation Xist RNA robustly returns to the Xi. One factor necessary for the localization of Xist RNA to the Xi after B cell stimulation is the transcription factor Yy1, as loss of Yy1 abrogates the localization of Xist RNA to the Xi in stimulated B cells. Yy1 was found to interact with multiple structural proteins in stimulated female B cells, but whether these factors cooperate to regulate XCI is unclear. Additionally, how the dynamic movement of Xist RNA affects the nuclear organization of the Xi is unknown. Using female follicular B cells, we will interrogate the nuclear organization of the Xi, and determine how Yy1-interacting structural proteins contribute to dynamic XCI maintenance. In Aim 1 we will perform allele-specific imaging of the Xi in nave and stimulated B cells to determine how dynamic movement of Xist RNA impacts organization of the Xi territory. We will examine compaction of the Xi, localization of the Xi territory within the nucleus, and organization of the Xi bipartite structure. We have recently found novel X-linked gene transcription from the Xi in female B cells, and will use this to generate gene-specific probes to examine spatial localization of these genomic loci within the Xi territory. We hypothesize that the dynamic movement of Xist RNA upon B cell stimulation will change the organization of the Xi territory. In Aim 2, we will determine the role of three Yy1-interacting structural proteins, LaminB1, Satb1, and condensins in XCI. Use of floxed mice to perform individual ex vivo deletions of each gene will determine their requirement for Xist RNA localization to the Xi, enrichment of canonical Xi heterochromatin marks H3K27me3 and H2AK119Ub, X-linked gene expression, and nuclear organization of the Xi territory. We hypothesize that these proteins cooperate with Yy1 to maintain XCI through regulation of Xist RNA localization and Xi nuclear organization in stimulated female follicular B cells. Together, these aims will expand our knowledge of the mechanisms regulating XCI maintenance, and contribute to our understanding of nuclear organization and gene regulation.